呼吸道合胞病毒M2：81-95与热休克蛋白70L1融合蛋白的制备及其免疫原性  

Construction and immunogenicity of fusion protein containing respiratory syncytial virus M2：81-95 and heat shock protein 70L1

作　　者：曾瑞红[1] 梅兴国[2] 亓晓温[2] 张振亚[2] 魏林[1] 

机构地区：[1]河北医科大学免疫教研室,石家庄050017 [2]军事医学科学院毒物药物研究所

出　　处：《中华传染病杂志》2009年第1期11-15,共5页Chinese Journal of Infectious Diseases

摘　　要：目的构建呼吸道合胞病毒（RSV）CTL表位M2：81-95与热休克蛋白（HSP）70L1融合蛋白的重组质粒，原核表达后初步研究其免疫原性。方法从SMMC7721细胞中克隆HSP70L1基因，PCR扩增M2：81-95基因片段，鉴定后构建质粒pET-HSP70L1-M2：81-95（pET-HSP70L1-M2），经PCR和酶切鉴定，转化E．coli BL21（DE3），用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达HSP70L1-M2：81-95（HSP70L1-M2），Ni^＋螯合亲合层析纯化，梯度透析复性。用该蛋白免疫10只BALB／c小鼠，ELISA检测IgG抗体及其亚型，噻唑蓝（MTT）法检测CTL杀伤活性。结果成功构建重组质粒pET-HSP70L1-M2，并在E．coli中表达重组蛋白HSP70L1-M2，该蛋白诱导RSV及表位特异性的CTL活性，还诱导蛋白抗原特异性的IgG，为4．87±0．35、IgG1为5．53±0．28、IgG2a为4．40±0．21，且IgG1／IgG2a（1．26）的比例平衡，与PBS对照组的IgG，为0．33±0．17、IgG1为0．51±0．21、IgG2a为0比较，差异有统计学意义（t=3．512，3．681，5．856；P＜0．05）。结论成功构建原核表达质粒pET-HSP70L1-M2，并表达纯化获得重组蛋白，免疫小鼠后诱导RSV及表位特异性的CTL活性和辅助性T淋巴细胞（Th）1／Th2混合型应答。Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus （RSV） cytotoxicity T lymphocyte （CTL） epitope M2：81-95 and heat shock protein （HSP） 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSPTOL1 gene was cloned from SMMC7721 cells. The M2：81-95 gene fragment was amplified by polymerase chain reaction （PCR） method. Plasmid pET HSPTOL1-M2：81-95 （pET-HSPTOL1- M2） was constructed, identified and transferred into E. coli BL21 （DE3）. Expression of HSPTOL1 M2：81-95（HSP70L1-M2） was induced by isopropy-β-D-thiogalactoside （IPTG）. The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme linked immunosorbent assay （ELISA） , and CTL responses were determined by methyl thiazolyl tetrazolium （MTT）. Results The recombinant plasmid pET-HSPTOL1-M2 was successfully constructed. The fusion protein HSPTOL1-M2 was expressed in E. coli. The purified protein induced strong RSV and CTL epitope-specific CTL responses and high titer of protein specific IgG antibody 4.87±0.35. The subtypes were IgG1 （5. 534±0.28） and IgG2a （4.40±0.21） and IgG1/IgG2a ratio was balanced. The titers of IgG, IgG1 and IgG2a in PBS control group were 0.33 ±0.17, 0.51±0.21 and 0, respectively, which were significantly lower than those in immunized group （t=3. 512, 3. 681, 5. 856; P〈0.05）. Conclusions The recombinant plasmid pET HSPTOL1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP701.1-M2 induced strong RSV-and CTL epitopespecific CTL responses and mixed T helper cell （Th）1/Th2 response in BALB/c mice.

关 键 词：呼吸道合胞病毒 T淋巴细胞 细胞毒性 表位 T淋巴细胞 热休克蛋白质类 重组融合蛋白质类 T淋巴细胞亚群 

分 类 号：R3[医药卫生—基础医学]